Method for pretreatment and method for analysis of lenalidomide in biological sample

ABSTRACT

The present invention addresses the problem of providing a novel method for the pretreatment of a biological sample containing lenalidomide enantiomer and thereby establishing a simple and accurate method for the quantitative analysis of lenalidomide enantiomer. In the present invention, the racemization and decomposition of lenalidomide enantiomer in a biological sample can be prevented by the deproteinization under acidic conditions of the biological sample containing lenalidomide enantiomer, and the lenalidomide enantiomer can be simply and accurately quantitatively analyzed by subjecting to HPLC the biological sample that has been pretreated in such a way.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a method for pretreatment of biologicalsamples containing enantiomers of lenalidomide, wherein deproteinizationis carried out under acidic conditions, and to a method for quantifyingenantiomers of lenalidomide in a biological sample, wherein a biologicalsample containing enantiomers of lenalidomide, pretreated in such amanner, is analyzed by high-performance liquid chromatography.

Related Background Art

Lenalidomide, a derivative of thalidomide, is widely used as aneffective immunomodulating drug for different malignant blood diseasessuch as multiple myeloma. Lenalidomide has been reported to have asuperior toxicity profile and more excellent immunomodulatory activitycompared to thalidomide (NPL 1). The pharmacokinetic properties oflenalidomide have been elucidated by HPLC methods in reversed-phase mode(NPL 2, NPL 3 and NPL 4), and the final excretion half-life oflenalidomide is conjectured to be about 3 to 4 hours.

In regard to thalidomide and its derivatives and analogs, differences indrug activity have been reported between the R-form and S-form. Forexample, the sedative action of thalidomide has only been reported withthe R-form, (NPL 5), while S-pomalidomide(3-amino-phthalimide-glutarimide) has been reported to significantlyinhibit corneal vascularization elicited by bFGF or VEGF, compared tothe R-form or racemic form (NPL 6).

Despite such differences in activity between the enantiomers,thalidomide is still administered as a racemic mixture withR-form:S-form=50:50. One of the main reasons for this is its property ofvery rapid racemization in blood (NPL 7).

The enantiomers of thalidomide have been separated and quantified byrepeated extraction using organic solvents, or by chromatographicmethods using an enantioselective stationary phase such as modifiedamylose (NPL 8), cellulose (NPL 7), vancomycin (NPL 9) or methacrylamide(NPL 10), and it has been shown that racemization is very rapid in theblood. Such racemization was first disclosed by G. Blaschke et al., whoreported that the racemization half-life of thalidomide in human bloodplasma is approximately 10 minutes (NPL 10). This is extremely shortconsidering that the excretion half-life of thalidomide is 8.7 hours(NPL 11).

Because it has such pharmacokinetic properties, it is thought that thereis no pharmacological significance in administering a pure enantiomer ofthalidomide, and it has been assumed that lenalidomide, which has abasic backbone similar to thalidomide, would also have similarproperties. Therefore, the pharmacokinetic and pharmacologicalproperties of the pure enantiomers of lenalidomide have not beenthoroughly researched, and absolutely no data has been reported onseparation and quantification of pure enantiomers of lenalidomide inbiological samples.

CITATION LIST Non-Patent Literature

-   [NPL 1] Hideshima T et al., Ther. Clin. Risk. Manag. 2008, 4(1), p.    129-36-   [NPL 2] Tohnya T M et al., J. Chromatogr. B Analyt. Technol. Biomed.    Life. Sci., 2004, 811(2), p. 135-41-   [NPL 3] Chen N et al., Cancer. Chemother. Pharmacol., 2012,    70(5), p. 717-25-   [NPL 4] Chen N et al., J Clin Pharmacol., 2007, 47(12), p. 1466-75-   [NPL 5] Hoeglund P et al., J. Pharmacokinet. Biopharm., 1998    26(4), p. 363-83-   [NPL 6] Lentzsch S et al., Cancer. Res., 2002, 62(8), p. 2300-5-   [NPL 7] Eriksson T et al., Chirality., 1995, 7(1), p. 44-52-   [NPL 8] Meyring M et al., J. Chromatogr. A. 2000, 876(1-2), p.    157-67-   [NPL 9] Murphy-Poulton S F et al., J. Chromatogr. B Analyt. Technol.    Biomed. Life. Sci., 2006, 831(1-2), p. 48-56-   [NPL 10] Knoche B et al., J. Chromatogr. A., 1994, 666, p. 235-240-   [NPL 11] Chen T L et al., Drug. Metab. Dispos., 1989, 17(4), p.    402-5

SUMMARY OF THE INVENTION

It is an object of the invention to provide a novel method forpretreatment of a sample that minimizes racemization and/ordecomposition of enantiomers of lenalidomide, maintaining the respectivecontents of the lenalidomide enantiomers as in the body.

The present inventors have found, surprisingly, that enantiomers oflenalidomide reside in the body for a significantly longer time thanenantiomers of thalidomide. As mentioned above, it has been assumed thatthere is no pharmacological significance in administering pureenantiomers of thalidomide because the enantiomers would have a veryrapid racemization rate in vivo, but it has been demonstrated thatlenalidomide resides in the body as enantiomers for a long period with anon-negligible pharmacological effect, considering its excretionhalf-life. Evaluation of the pharmacokinetic and pharmacological actionof pure enantiomers of lenalidomide therefore potentially has veryimportant significance. When pure enantiomers of lenalidomide in abiological sample are to be analyzed, it is necessary to conductdeproteinizing treatment beforehand in order to prevent reduction inanalysis precision due to interference by proteins. Pretreatment ofthalidomide has conventionally been carried out by extracting athalidomide-containing biological sample with a strongly hydrophobicorganic solvent, either once or several times, drying the obtainedorganic solvent layer, and then redissolving it in an organic solvent.Lenalidomide, however, has higher polarity than thalidomide and lowersolubility in the organic solvent layer, such that the conventionalmethod has not been applicable. In order to solve this problem, thepresent inventor conducted much diligent research on the effect of pH onracemization of lenalidomide enantiomers, and as a result have foundthat enantiomers of lenalidomide are highly stable under acidicconditions, and the invention has thus been completed.

Specifically, the invention encompasses the following inventions.

[1] A method for pretreatment of a biological sample containingenantiomers of lenalidomide, wherein deproteinization of the biologicalsample is carried out under acidic conditions.

[2] The method according to [1], wherein the acidic conditions are pH 5or lower.

[3] The method according to [1] or [2], wherein an acid selected fromamong perchloric acid, trichloroacetic acid, trifluoroacetic acid,metaphosphoric acid, hydrochloric acid, succinic acid and maleic acid isadded.

[4] The method according to any one of [1] to [3], wherein thebiological sample is blood, serum, blood plasma, urine, saliva, breastmilk, spinal fluid, semen, tissue or microsomes.

[5] The method according to any one of [1] to [4], wherein theenantiomer of lenalidomide is the S-form.

[6] The method according to any one of [1] to [4], wherein theenantiomer of lenalidomide is the R-form.

[7] The method for quantifying enantiomers of lenalidomide in abiological sample, wherein a biological sample containing enantiomers oflenalidomide that has been pretreated by a method according to any oneof [1] to [6] is separated and analyzed by high-performance liquidchromatography.

[8] A method for storing enantiomers of lenalidomide, wherein theenantiomers of lenalidomide are kept under acidic conditions in anaqueous buffer.

[9] The method according to [8], wherein the acidic conditions are pH 5or lower.

[10] The method according to [8] or [9], wherein the aqueous buffer iscitrate buffer.

[11] The method according to any one of [8] to [10], wherein theenantiomer of lenalidomide is the S-form.

[12] The method according to any one of [8] to [10], wherein theenantiomer of lenalidomide is the R-form.

According to the invention it is possible to pretreat a sample whileminimizing racemization and/or decomposition of enantiomers oflenalidomide and maintaining the enantiomer contents of lenalidomide asin the body, to conveniently and efficiently separate and quantifyenantiomers of lenalidomide in a biological sample. This allows thepharmacokinetic properties or pharmacological properties of the pureenantiomers of lenalidomide in the body to be elucidated, to potentiallyaid in discovering possibilities for reducing side-effects or increasingdrug effects by administration of the pure enantiomers of lenalidomide.The invention will therefore definitely provide a major contribution toapplication and development in the medical and pharmaceuticalindustries, including research on the pharmacological drug effects,safety, physical properties and pharmacokinetics of enantiomers oflenalidomide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of graphs showing the stability of lenalidomide storedat room temperature and at 50° C. for 24 hours, in different solvents.The solid black bars represent enantiomer 1, and the diagonally shadedbars represent enantiomer 2.

FIG. 2 shows separation of lenalidomide enantiomers using Chiralpak IC(4.6 mm i.d.×250 mm) as the optical resolution column, with ethylacetate as the mobile phase.

FIG. 3 shows purification of lenalidomide enantiomers with Chiralpak IC(20 mm i.d.×250 mm) as the optical resolution column, and with ethylacetate as the mobile phase. The enantiomer eluted in fraction 1 wasdesignated as LL1, and the enantiomer eluted in fraction 2 wasdesignated as LL2.

FIG. 4 is a set of chromatograms of purified (fractionated) enantiomersin fraction 1 (A-1) and fraction 2 (B-1). Chiralpak IA was used for theanalysis.

FIG. 5 is a chromatogram showing S-thalidomide (a) and R-thalidomide(b).

FIG. 6 shows time-dependent changes in the peak area of (S*)-LL and(R*)-LL after adding aqueous buffer at room temperature.

FIG. 7 shows time-dependent changes in enantiomer excess in aqueousbuffers (pH 4 to 9) at room temperature.

FIG. 8 is a plot of log₁₀(Enantiomer Excess) with respect to time, atroom temperature.

FIG. 9 is a pair of chromatograms of (a) LL enantiomer in water(control) and (b) LL enantiomer in human serum. Sample: (a) 0.1 mg/mlracemic LL in water, (b) 0.1 mg/ml racemic LL in human serum, (c)LL-free human serum. HclO₄ was added immediately after dissolving the LLin serum or water. HPLC conditions: Column: Chiralpak IA (4.6 mmi.d.×250 cm)+Security guard C8 (3.0 mm i.d.×4 mm), flow rate: 0.75mL/min, injection rate: 2 μL.

FIG. 10 is a set of chromatograms of (a) 0.01 mg/mL LL in Na-phosphoratebuffer (pH 7.4), (b) 0.01 mg/mL LL in serum, and (c) LL-free blankserum.

FIG. 11 is a chromatogram for human serum at 37° C., after adding 10μg/mL of (S*)-LL.

FIG. 12 shows the racemization half-lives of LL and TD enantiomers inNa-phosphorate buffer (pH 7.4, diagonal-shaded bars) and human serum(solid black bars). The bar graphs represent the average values for 3tests, and the error bars represent SD. The significant differences were**p<0.01 and ***p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

Lenalidomide (LL) is a derivative of thalidomide (TD) which is known asa therapeutic agent for multiple myeloma, and it is widely used as aneffective immunomodulating drug for different malignant blood diseasessuch as multiple myeloma. It has the chemical structure represented bythe following structural formula, having an asymmetric center on adioxopiperidine ring, similar to thalidomide.

When used in combination with dexamethasone, lenalidomide has a highsuccess rate of 60% for recurrent intractable multiple myeloma, and itis receiving approval throughout the world, having been approved inabout 50 countries so far, and has become a blockbuster drug with salesexceeding 400 billion yen. It was approved in 2010 in Japan, but withthe condition of compliance with administrative procedures by medicalpersonnel, patients and family, from the viewpoint of ensuring safety,as well as the requirement to investigate the merit of its use in allcases. Lenalidomide has a greater drug effect than thalidomide and fewerside-effects such as sleepiness and numbness, but also, likethalidomide, carries the risk of teratogenicity, and therefore its useis contraindicated for pregnancy. Because lenalidomide is a derivativeof thalidomide, it is also expected to racemize rapidly in the blood inthe same manner as thalidomide. It is therefore sold as anR-form:S-form=50:50 racemic mixture, and it is currently difficult toobtain pure enantiomers of lenalidomide. Furthermore, since analysisconditions for controlling racemization and accomplishing stablepretreatment and separation have not been established, virtually no dataexist regarding the pharmacokinetic properties and racemization rate ofpure enantiomers of lenalidomide.

The present inventors have found that it is possible to separate andpurify pure enantiomers of lenalidomide with controlled decompositionand racemization, by using an organic solvent selected from the groupconsisting of aprotic solvents, secondary alcohols and mixtures thereofas the mobile phase, for optical resolution by chromatography. Accordingto one mode of the invention, therefore, there is provided a method forseparating and purifying lenalidomide enantiomers, wherein a samplecontaining an enantiomeric mixture of lenalidomide is supplied forchromatography, and an organic solvent selected from the groupconsisting of aprotic solvents, secondary alcohols and mixtures thereofis used as the mobile phase for stable optical resolution of eachenantiomer of lenalidomide from an enantiomeric mixture of lenalidomide.

In the chromatographic method, a sample containing an enantiomericmixture in a stationary phase supporting a compound with asymmetricdiscriminatory power (a chiral discriminator) is supplied together withthe organic solvent as the mobile phase, adsorbing each enantiomer andutilizing their difference in retention time for optical resolution ofeach enantiomer. It is generally carried out using a high-performanceliquid chromatography (HPLC) apparatus comprising an optical resolutioncolumn.

An aprotic solvent to be used as the mobile phase is not particularlyrestricted so long as it allows the object of the invention to beachieved, and examples include esters such as acetonitrile and ethylacetate, ketones such as acetone, ethers such as diethyl ether anddiisopropyl ether, and combinations of the foregoing. Preferred areacetonitrile, ethyl acetate and combinations thereof.

A secondary alcohol to be used as the mobile phase is not particularlyrestricted so long as it allows the object of the invention to beachieved, and examples include isopropanol, 2-butanol, cyclopentanol andcyclohexanol, and combinations of the foregoing. Isopropanol ispreferred among these.

The optical resolution column to be used for the method of the inventionis not particularly restricted so long as it allows the object of theinvention to be achieved, and it may be a normal-phase column orreversed-phase column, or a separation mode column or a multimode columncomprising a combination of these. A polysaccharide derivative chiralcolumn will typically be used. A polysaccharide derivative chiral columnis a column in which a polysaccharide derivative as the chiraldiscriminator is immobilized on a support. The polysaccharide derivativesupported on the polysaccharide derivative chiral column may be anamylose derivative or cellulose derivative, for example. According tothe invention, it is preferred to use Chiralpak™ IA or Chiralpak™ IC.

The optical resolution method of the invention allows pure enantiomersof lenalidomide to be separated and purified from an enantiomericmixture (such as a racemic mixture) of lenalidomide, withoutdecomposition or racemization.

When pure enantiomers of lenalidomide in a biological sample are to beseparated and purified, the macromolecules such as proteins that canlower the low-molecular separation efficiency are generally removedbeforehand. Pretreatment of thalidomide has conventionally been carriedout by extracting a thalidomide-containing biological sample with ahydrophobic organic solvent (such as an n-hexane/ethyl acetate mixture),either once or several times, drying the obtained organic solvent layer,and then redissolving it in an organic solvent such as dioxane.Lenalidomide, however, has higher polarity than thalidomide and lowersolubility in the organic solvent layer, such that the conventionalmethod has had poor efficiency and has been inadequate. In order tosolve this problem, the present inventors conducted much diligentresearch on the effect of pH on racemization of lenalidomideenantiomers, and as a result have found that enantiomers of lenalidomideare highly stable under acidic conditions. According to another mode ofthe invention, therefore, there is provided a method for pretreatment ofa biological sample containing enantiomers of lenalidomide, wherein thebiological sample is deproteinized under acidic conditions.

Such acidic conditions are typically pH 5 or lower, preferably in therange of pH 2 to pH 5, and most preferably in the range of pH 4 to pH 5.

The acid to be added to the sample to produce the acidic conditions isnot particularly restricted so long as it allows the object of theinvention to be achieved, and may be perchloric acid, trichloroaceticacid, trifluoroacetic acid, metaphosphoric acid or hydrochloric acid, ora dicarboxylic acid such as succinic acid or maleic acid.

The biological sample is not particularly restricted so long as itcontains lenalidomide, and examples include blood, serum, blood plasma,urine, saliva, breast milk, sweat, spinal fluid, semen, tissue andmicrosomes.

By supplying a pretreated biological sample containing lenalidomideenantiomers by chromatography, and preferably high-performance liquidchromatography (HPLC), it is possible to conveniently and efficientlyseparate and quantify enantiomers of lenalidomide in the biologicalsample.

Moreover, by keeping the fractionated lenalidomide enantiomers in anaqueous buffer under acidic conditions, it is possible to store thelenalidomide enantiomers. Such acidic conditions are typically pH 5 orlower, and preferably in the range of pH 2 to pH 5. The aqueous bufferis not particularly restricted so long as it allows the acidicconditions to be maintained, and examples include citrate buffer,phosphate buffer, acetate buffer, glycine-hydrochloride buffer,MES-HEPES buffer, Tris buffer and borate buffer.

EXAMPLES Example 1. Stability of Lenalidomide in Different Solvents

A racemic mixture of 0.5 mg/mL lenalidomide in methanol, ethanol,isopropanol, acetonitrile or ethyl acetate, as the sample (product ofSelleck Chemicals, US) was incubated at room temperature or at 50° C.for 24 hours, and the following HPLC conditions were employed forquantification of the lenalidomide enantiomers, for evaluation of thestability of lenalidomide in the different solvents.

HPLC Conditions Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

Column: Chiralpak IA (4.6×250 cm, 5 μm, product of Dicel), RTMobile phase: EtOH (100%)Flow rate: 1.0 mL/minInjection volume: 5 μL

Detection: UV 230 nm

The peak areas for the enantiomers before and after incubation are shownin FIG. 1 (the initially observed peaks are enantiomer 1, and the nextobserved peaks are enantiomer 2). Lenalidomide was highly unstable inmethanol and ethanol, but no decomposition was observed in acetonitrileand ethyl acetate. Lenalidomide was also stable even in isopropanol.Considering these results, it is thought that decomposition oflenalidomide is accelerated by the weak basicity of the alcohol thatattacks the α-carbon atom of the carbonyl group. This does notcontradict the results indicating stability of lenalidomide inacetonitrile and ethyl acetate, which are aprotic solvents.

Example 2. Separation of Lenalidomide Enantiomers Using DifferentSolvents

In order to elucidate the racemization rate of enantiomers oflenalidomide it is very important to separate and quantify the pureenantiomers of lenalidomide. As demonstrated in Example 1, lenalidomidein an alcohol (especially methanol and ethanol) or water (>pH 7) solventis unstable, and therefore a highly stable aprotic solvent should beused as the mobile phase or sample solvent.

Separation of enantiomers of lenalidomide (LL) and thalidomide (TD) wastested using the different organic solvents listed in Table 1 as themobile phase, under the following HPLC conditions. The samples used were0.5 mg/mL LL (Wako Pure Chemical Industries, Ltd.) and TD (Sigma-AldrichJapan, KK.) dissolved in acetonitrile.

HPLC Conditions Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

Column: Chiralpak IC (4.6×250 cm, 5 μm, product of Dicel), RTMobile phase: Solvent listed in Table 1Flow rate: 1.0 mL/minInjection volume: 5 μL

Detection: UV 230 nm or 254 nm

The mobile phase used was an aprotic solvent comprising acetonitrile(ACN), ethyl acetate (EtOAc), tetrahydrofuran (THF) and t-butyl methylether (BME). Isopropanol (IPA) is a secondary alcohol, and it was alsotested because it has excellent stability, as shown in FIG. 1. Theenantiomer separating power was evaluated using the separation factor(RS), defined by the following formula.

RS=2×(T ₂ −T ₁)/(W ₁ +W ₂)

In the formula, numeral 1 and numeral 2 refer to enantiomer 1 andenantiomer 2, respectively (peak 1 eluting before peak 2), T representsthe retention time, and W represents the peak width. Table 1 shows theseparation of LL enantiomers when using different solvents as the mobilephase.

TABLE 1 Enantiomer separation using Chiralpak IC with different inertmobile phases Method Mobile phase composition (%) Separation No. ACNEtOAc THF IPA tBu—O—Me LL TD 01 100 7.26 2.51 02 100 12.51 0.59 03 1002.93 NS 04 75 25 6.33 1.08 05 75 25 2.05 NS 06 75 25 4.78 NS 07 50 506.40 NS 08 50 50 NS NS 09 50 50 4.51 NS 10 50 50 4.90 NS 11 50 50 NS NS12 50 25 25 4.04 NS 13 50 50 1.53 NS 14 50 25 25 2.45 1.43 15 25 75 7.621.84 16 75 25 8.24 1.35 17 75 25 10.11 NS 18 100 NS NS NS: No separation

Among these mobile phases, the most effective ones for separation of LLenantiomers was ethyl acetate, the highest separation being obtainedwhen using ethyl acetate as the mobile phase (Table 1, No. 02)(RS=12.51). FIG. 2 shows that LL enantiomers were satisfactorilyseparated when using ethyl acetate as the mobile phase. Moreover, usinga mobile phase with a fixed amount of ethyl acetate mixed exhibited moreexcellent separation than when no ethyl acetate was present or theamount was very low (for example, No. 3, 10, 11 and 17). These resultsdemonstrated that ethyl acetate is a highly superior solvent as themobile phase in chromatography for LL enantiomer separation.

Acetonitrile also exhibited satisfactory separation (Table 1, No. 01,RS=7.26). Since acetonitrile also exhibits excellent stability as shownin FIG. 1, it can serve as a satisfactory solvent for the mobile phasein chromatography for separation of LL enantiomers.

For TD, on the other hand, sufficient separation was not observed whenusing any solvent as the mobile phase.

Example 3. Preparation of Pure Enantiomers of Lenalidomide

A method for preparing pure enantiomers of lenalidomide was established,based on the results for stability and enantiomer separation oflenalidomide. Specifically, pure enantiomers of lenalidomide wereprepared according to the following scheme and HPLC conditions.

HPLC Conditions Apparatus: HPD PUMP and LAMBDA1010 (Bischoff Co.)

Column: Chiralpak IC (2.0 mm i.d.×250 cm, 5 μm, product of Dicel), RTMobile phase: Ethyl acetateFlow rate: 10 mL/minInjection volume: 2.3 μL/mL LL racemic mixture in 5000 μL ofacetonitrile

Detection: UV 254 nm

As shown in FIG. 3, the LL enantiomers were completely separated. Afterseparating off the fractions and drying them, over 80 mg of the pureenantiomers was obtained from 180 mg of the racemic mixture. Thefractions were dissolved in acetonitrile (1 or 0.1 mg/mL) and stored at−25° C. Based on the integral peak areas in the chromatograms shown inFIG. 4 (A-2, B-2), fraction 1 and fraction 2 recovered as lenalidomideenantiomer 1 (LL1) and lenalidomide enantiomer 2 (LL2) exhibitedsufficient enantiomer purities of 99.02% and 99.96%, respectively.

Example 4. Inference of Absolute Configurations of LL1 and LL2 fromElution Order

The absolute configurations of LL1 and LL2 can be reasonably inferred bycomparing the order of elution of LL1 and LL2 with commerciallyavailable R-thalidomide and S-thalidomide (Sigma-Aldrich Japan, KK.).The order of elution was confirmed with the following HPLC conditions.

HPLC Conditions Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

Column: Chiralpak IA (4.6 mm i.d.×250 cm, 5 μm, product of Dicel), RTMobile phase: 0.1% Formic acid in EtOH/H₂O (95/5, v/v)Flow rate: 0.75 mL/minInjection volume: 5 μL of 0.1 mg/mL (S)-TD, (R)-TD

Detection: UV 230 nm

As clearly seen from FIG. 5, S-thalidomide eluted earlier thanR-thalidomide. Considering that LL1 elutes earlier than LL2 under thesame HPLC conditions, presumably the absolute configuration of LL1corresponds to the S-enantiomer while the absolute configuration of LL2corresponds to the R-enantiomer (hereunder, LL1 and

LL2 will be defined as S*-LL and R*-LL).

Example 5. Dependence of Racemization Half-Life on pH

In order to elucidate the racemization half-life of lenalidomide and itspH dependence, an experiment was conducted with the following scheme andHPLC conditions.

TABLE 4 pH Measured pH Buffer 4 3.98 0.05M Na-citrate buffer 5 5.00 66.02 7(a) 6.97 0.05M NH₄-acetate buffer 7(b) 7.04 0.05M Na-phosphatebuffer 8 7.96 0.05M Tris-HCl buffer 9 9.03

HPLC Conditions Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

Column: Chiralpak IA (4.6 mm i.d.×250 cm, 5 μm, product of Dicel), RTMobile phase: 0.1% Formic acid in EtOH/H₂O (95/5, v/v)Flow rate: 0.75 mL/min

Injection: 5 μL Detection: UV 230 nm

The variation in (S*)-LL at different pH is shown in FIG. 6. A smallamount of (R*)-LL was produced with incubation at pH 6, whileracemization was significantly accelerated at pH 7 and higher. Since thepeak areas of (S*)- and (R*)-LL were reduced at pH 9 and 8, it wasconcluded that racemization and decomposition of LL takes place underthese pH conditions.

The enantiomeric excess rate (EE, [(S−R)/(S+K)×100], %) at each point isplotted in FIG. 7, and the log₁₀(EE) vs. time plot is shown in FIG. 8.As reported for thalidomide, since racemization is thought to be apseudo-first-order reaction, FIG. 8 shows an approximately idealstraight line.

This linearity was used to calculate the time for EE=50 based on astraight line approximation (y=ax+b), to determine the racemizationhalf-life. The stability of the pure enantiomer is very highly dependenton pH, the half-life being 10 times longer for every pH reduction of 1,and estimation of the half-life was no longer possible at pH 4. At pH 9,on the other hand, sudden racemization and decomposition were observed.These results clearly indicate that enantiomers of lenalidomide arestable under acidic conditions (<pH 4).

Example 6. Establishing Biological Sample Pretreatment Method

Numerous reports already exist of measuring thalidomide or lenalidomidein blood (serum and blood plasma) or urine using HPLC. Measurement ofenantiomers of TD in biological samples has been reported, and theracemization half-life has been determined both in vivo and in vitro(Eriksson T et al., Chirality., 1995, 7(1), p. 44-52 and Knoche B etal., J. Chromatogr. A., 1994, 666, p. 235-240). These measurements areall based on liquid-liquid extraction methods. The conventional methodis shown as scheme (a). Measurement is possible by this method becausean aprotic solvent is essentially inert to racemization ordecomposition. However, since the solubility of thalidomide orlenalidomide is not high in hydrophobic organic solvents, it isnecessary to use a large amount of solvent and carry out repeatedextraction.

A very simple and efficient pretreatment method for lenalidomide wastherefore established, based on the knowledge of enantiomer stabilityunder acidic conditions demonstrated in Example 5. The analysis methodof the invention is shown as scheme (b). The acid used to suspend thereaction while simultaneously precipitating and removing the protein wasHClO₄.

FIG. 9 shows chromatograms obtained using the method of scheme (b). Asshown in FIG. 9(b), lenalidomide enantiomers in serum can besatisfactorily separated and quantified without being affected bycontaminating components such as proteins, and therefore the method canbe applied to biological samples as well.

Example 7. Racemization of Lenalidomide Under Physiological Conditions

The obtained pure enantiomers were used to evaluate the racemizationrate of lenalidomide under biological buffer conditions (pH 7.4, 37° C.)and in human serum (37° C.), by the following scheme.

HPLC Conditions Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

Column: Chiralpak IA (4.6 mm i.d.×250 cm, 5 μm)+Security guard C8 (3.0mm i.d.×4 mm)

Temperature: 40° C.

Mobile phase: 0.1% Formic acid in EtOH/H₂O (95/5, v/v)Flow rate: 0.75 mL/min

Injection: 20 μL Detection: UV 230 nm

As clearly seen from the chromatogram for (S*)-LL shown in FIG. 10 [0.01mg/mL (a) in buffer, (b) in serum], the method of the invention hassufficient retention and separation and detection sensitivity(signal-to-noise ratio). The blank sample (c) derived from serum alonewithout LL contained no contaminants that inhibit measurement of LL inthe method of the invention. The peak area for (S*)-LL in FIG. 18(b) was91% of FIG. 18(a). This indicates an adequate recovery rate forpretreatment of lenalidomide in serum.

FIG. 11 shows continuous change in the chromatogram for incubation ofS*-LL. The LL enantiomers were separated and quantified to a sufficientlevel for detection of racemization product, without being inhibited byother substances in the sample.

The racemization half-life was calculated by the same method describedin Example 5. The racemization half-life in aqueous buffer was estimatedfrom this, as follows: (S*)-LL, (R*)-LL, (S)-TD and (R)-TD=272±1.3,267±1.1, 266±10 and 295±24 (min), respectively. These resultsdemonstrated that at pH 7.4, LL enantiomers have a similar half-life asTD in aqueous buffer.

The half-life in human serum was calculated in the same manner: (S*)-LL,(R*)-LL, (S)-TD and (R)-TD=131±4.9, 109±2.0, 21.8±1.0 and 32.9±25 (min),respectively. The results are shown in FIG. 12. Thalidomide in humanserum has a half-life of only 8.2% (S-form) and 11.1% (R-form) comparedto buffer, indicating that racemization of thalidomide is extremelyaccelerated in serum. It is believed that the accelerated racemizationfor these enantiomers is due to the biomolecules including proteins suchas human albumin in serum.

For LL in serum, the results showed that (S*)-LL has a racemizationhalf-life of 6 times longer than (S)-TD, while (R*)-LL has aracemization half-life of 3 times longer than (R)-TD. These resultsclearly indicate that the LL enantiomers, and especially (S*)-LL, aremuch more stable compared to thalidomide. In contrast to TD, (S*)-LL wasmore stable than (R*)-LL. This difference in the half-lives of the S-and R-enantiomers should also be noted.

1. A method for pretreatment of a biological sample containingenantiomers of lenalidomide, wherein deproteinization of the biologicalsample is carried out under acidic conditions.
 2. The method accordingto claim 1, wherein the acidic conditions are pH 5 or lower.
 3. Themethod according to claim 1, wherein an acid selected from amongperchloric acid, trichloroacetic acid, trifluoroacetic acid,metaphosphoric acid, hydrochloric acid, succinic acid and maleic acid isadded.
 4. The method according to claim 1, wherein the biological sampleis blood, serum, blood plasma, urine, saliva, breast milk, spinal fluid,semen, tissue or microsomes.
 5. The method according to claim 1, whereinthe enantiomer of lenalidomide is the S-form.
 6. The method according toclaim 1, wherein the enantiomer of lenalidomide is the R-form.
 7. Themethod for quantifying enantiomers of lenalidomide in a biologicalsample, wherein a biological sample containing enantiomers oflenalidomide that has been pretreated by a method according to claim 1is separated and analyzed by high-performance liquid chromatography. 8.A method for storing enantiomers of lenalidomide, wherein theenantiomers of lenalidomide are kept under acidic conditions in anaqueous buffer.
 9. The method according to claim 8, wherein the acidicconditions are pH 5 or lower.
 10. The method according to claim 8,wherein the aqueous buffer is citrate buffer.
 11. The method accordingto claim 8, wherein the enantiomer of lenalidomide is the S-form. 12.The method according to claim 8, wherein the enantiomer of lenalidomideis the R-form.